Nonisotopic detection systems have become a preferred mode in scientific research and clinical diagnostics for the detection of biomolecules using various assays including, but not limited to, flow cytometry, nucleic acid hybridization, DNA sequencing, nucleic acid amplification, microarrays, immunoassays, histochemistry, and functional assays involving living cells. In particular, while fluorescent organic molecules such as fluorescein and phycoerythrin are used frequently in detection systems, there are disadvantages in using these molecules in combination. For example, each type of fluorescent molecule typically requires excitation with photons of a different wavelength as compared to that required for another type of fluorescent molecule. However, even when a single light source is used to provide a single excitation wavelength (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different fluorescent molecules to permit individual and quantitative detection without substantial spectral overlap.
Additionally, conventional fluorescent molecules have limited fluorescence intensity. Further, currently available nonisotopic detection systems typically are limited in sensitivity due to the finite number of nonisotopic molecules which can be used to label a biomolecule to be detected.
Doped metal oxide (“DMO”) nanocrystals are nanocrystals that can be excited with a single excitation light source resulting in a detectable fluorescence emission of high quantum yield (e. g., a single quantum dot having at a fluorescence intensity that may be a log or more greater than that a molecule of a conventional fluorescent dye) and with a discrete fluorescence peak. Typically, they have a substantially uniform size of less than 200 Angstroms, and preferably have a substantially uniform size in the range of sizes of from about 1 nm to about 5 nm, or less than 1 nm.
In that regard, dMO nanocrystals are preferably comprised of metal oxides doped with one or more rare earth elements, wherein the dopant comprising the rare earth element is capable of being excited (e. g., with ultraviolet light) to produce a narrow spectrum of fluorescence emission (typi-cally more narrow than the spectrum of fluorescence emission emitted by a semiconductor nanocrystal). Such dMO nano-crystals are well known in the art. However, a desirable feature of dMO nanocrystals when used for nonisotopic detection applications is that the nanocrystals be made water-soluble. “Water-soluble” is used herein to mean that the nanocrystals are sufficiently soluble or suspendable in an aqueous-based solution including, but not limited to, water, water-based solutions, and buffer solutions, that are used in detection processes, as known by those skilled in the diagnostic art.
Semiconductor nanocrystals are quantum dots that can be excited with a single excitation light source resulting in a detectable fluorescence emission of high quantum yield (e.g., a single quantum dot having at a fluorescence intensity that may be a log or more greater than that a molecule of a conventional fluorescent dye) and with a discrete fluorescence peak. Typically, they have a substantially uniform size of less than 200 Angstroms, and preferably have a substantially uniform size in the range of sizes of from about 1 nm to about 5 nm, or less than 1 nm. In that regard, quantum dots are preferably comprised of a Group II-VI semiconductor material (of which ZnS, and CdSe are illustrative examples), or a Group III-V semiconductor material (of which GaAs is an illustrative example). Such quantum dots are well known in the art. However, a desirable feature of quantum dots when used for nonisotopic detection applications is that the quantum dots be made water-soluble. Current methods of making semiconductor nanocrystals water-soluble is to add to the semiconductor nanocrystal a layer comprising mercaptocarboxylic acid (Chen and Nie, 1998, Science 281:2016–2018), or silica (U.S. Pat. No. 5,990,479), or one or more layers of amino acids (U.S. Pat. No. 6,114,038). Depending on which layer composition is used, the treated nanocrystal may have limited stability in an aqueous solution, particularly when exposed to air (oxygen) and/or light. More particularly, oxygen and light can cause the molecules comprising the layer to become oxidized, thereby forming disulfides which destabilize the attachment of the layer molecules to the semiconductor nanocrystals. Thus, oxidation may cause the layer molecules to become detached from the surface of the quantum dots, there-by exposing the surface of the quantum dots which may result in “destabilized quantum dots”. Destabilized quantum dots form aggregates when they interact together, and the formation of such aggregates eventually leads to irreversible flocculation of the quantum dots. Additionally, depending on the layer composition, it can cause non-specific binding, particularly to one or more molecules in a sample other than the target molecule, which is not desirable in a detection assay.
Hence, there is a need to provide alternative forms of water-soluble, fluorescent nanocrystals.